Human carcinoembryonic antigen (CEA), which is extensively expressed in the majority of human cancers is a potential target for specific immunotherapy using recombinant vaccines. Following immunizations of recombinant vaccinia-CEA (rV-CEA), enhancement of CEA specific cytotoxic T lymphoctyes (CTL) responses was observed in patients. These studies demonstrated significant difference in the precursor frequency of anti- tumor CTL pre- vs post-immunization. A CEA-specific T-cell line was established from a patient with metastatic colorectal carcinoma and cultured long-term in vitro. The cell line (designated V8T) was shown to be CD8+, CD2+/CD54+, CD45+/CD49d+, and CD11a+/CD58+. V8T cells lyse CAP-1 pulsed C1R:A2 cells. V8T did not lyse non-HLA-A2,CEA positive allogeneic carcinoma cells. Allogeneic HLA-A2 positive and CEA negative carcinoma cell could not serve as target for V8T cells while an allogeneic HLA-A2 and CEA positive carcinoma cell line was lysed. The cytotoxicity of V8T cell line was shown to be MHC class I restricted. V8T long-term cultures were analyzed to ascertain the feasibility of in vitro expansion of CEA specific T cell lines for potential adoptive transfer. The Vb gene usage, production of cytokines, expression of homing-associated molecules (CD11a,CD49d) and cytotoxic activity of V8T in long-term cultures were evaluated. Results on the cytokine production, expression of homing and adhesion-associated molecules and CTL activity of V8T demonstrate stability of these phenotypes through long term passage. An additional CEA specific T cell line was established using CAP-1 peptide, from a stomach cancer patient enrolled in a Phase I recombinant avipox virus-CEA (ALVAC-CEA) clinical trial. At the same time, we also have access to the autologous tumor from the patient. An autologous tumor cell line was established and was used as a target in a CTL assay. GTn#11 T cells lyse autologous tumor cells (GTn#1 tumor) and allogeneic tumor cells as well as CAP-1 pulsed C1R:A2. Both HLA-A2 and CEA were expressed on GTn#11 tumor cells. In this study we have demonstrated that CTL generated using CAP-1 peptide can kill not only CAP-1 pulsed C1R:A2 cells but also autologous tumor cells. The ability of other HLA-A2 binding peptides, in addition to CAP-1 peptide to induce CEA specific CTL will be investigated. We have recently initiated a study using the CAP-2 peptide. CAP-2 is a 9 amino acid peptide (position 555-563 in CEA). PBMCs obtained from Vac8 patient were analyzed for T cell responses to CAP-2. Two cytotoxic T cell lines were established and both T cell lines lyse CAP-2 pulsed C1R:A2 cells and CEA positive, HLA-A2 positive carcinoma cells in a HLA-A2 restricted manner. We also investigated the ability of prostate specific antigen (PSA) epitopes to activate human cytotoxic T lymphocytes in vitro. Two novel HLA-A2 binding PSA epitopes (10 mers, designated PSA-1 and PSA-3) have been identified. Four T-cell lines were established from the peripheral blood lymphocytes of three different HLA-A2 donors by incubation of the PBMCs with PSA-1 or PSA-3 and IL-2. Six to seven IVS cycles were required for the generation of these PSA-peptide specific T-cell lines. T-cell lines TA-PSA-3(T-cell line established from donor A using peptide PSA-3), TB-PSA-1 (T-cell line established from donor B using peptide PSA-1), and TB-PSA-3 (T-cell line established from donor B using peptide PSA-3) were established from normal donors, whereas TP-PSA-3 (T-cell line established from donor P using peptide PSA-3) was established from a prostate cancer patient. All four T-cell lines were cytotoxic to T2 cells that had been pulsed with the corresponding PSA-1 or PSA-3 peptides that were used to establish them. All four T cell lines were shown to lyse LNCaP cells (HLA-A2+, PSA positive prostate cancer cells) (Table 10). The lysis of LNCaP did not involve natural killer cells. Addition of unlabeled LNCaP cells decreased the CTL activity against T2 cells pulsed with peptide PSA-3 when TA-PSA-3 or TB-PSA-3 cells were used as effectors. A novel HLA-A3 binding PSA epitope (9-mer, designated PSA-9) and a longer PSA peptide (30 mer, designated PSA-OP), containing PSA-1, PSA-3 and PSA-9 peptide sequences have also been identified. CTLs were established, and the specificity of these cell lines to stimulating peptide was determined by cytotoxic assays using peptide pulsed C1R:A2 or C1R:A3 cells. The PSA-OP specific CTLs lysed the HLA-A2 and PSA positive LNCaP prostate carcinoma cells. These studies demonstrate for the first time the ability of human peptides to be capable of generation of human CTL lines, and the ability of human prostate carcinoma cells to endogenously process PSA to present specific PSA peptides in the context of MHC class I for T-cell lysis. We have also demonstrated that it is possible toinduce CTL response in HLA-A2 transgenic mice by imunization with PSA peptide with adjuvant. Recognition by CTLs suggest that PSA peptides may be a potential candidate for use in peptide-based vaccines for prostate cancers.